Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH₃, Se, and NH

Abstract

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an a-b CH 2 group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the b-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the b-phosphate with BH 3 or Se, or substituted at either the a-b or b-g O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m 2 7,29-O Gpp S pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m 7 Gpp BH3 pm 7 G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t 1/2 ffi 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.

mRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

“…HU added immediately after nucleoporation accelerated the decay of both types of Not determined in the current work. The translational efficiencies of BTH-Luc-A 60 and nine other mRNAs in the A 60 series were reported in an earlier study (Su et al 2011).…”

“…These were introduced into HeLa cells by nucleoporation, and the loss of sequences over time was followed by qRT-PCR. We have used this approach to introduce mRNAs into cultured mammalian cells previously and have validated the qRT-PCR method of quantifying mRNA by two alternative methods: Northern blotting and autoradiography of 32 P-labeled mRNA (Grudzien et al 2006;Grudzien-Nogalska et al 2007a;Su et al 2011). The ARCA (anti-reverse cap analog) (Fig.…”

“…1A) has the same characteristics as the natural cap with respect to translation and hydrolysis by hDcp2, but it has a modification at the C29 position of the m 7 Guo moiety to prevent it from being incorporated into mRNA in the reverse orientation by T7 polymerase (Stepinski et al 2001;Jemielity et al 2003). mRNAs capped with the BTH analog (borano two-headed) are more efficiently translated than those capped with the ARCA and are completely resistant to hDcp2 hydrolysis in vitro (Su et al 2011).…”

“…When decapping cannot occur (with BTH-Luc-SL), the rapid-decay phase is delayed, but the mRNA is eventually degraded by other processes. However, just because BTH is resistant to hDcp2 (Su et al 2011), it does not mean that hDcp2 is responsible for decapping of ARCA-Luc-SL, since there are other mammalian decapping enzymes (Xiang et al 2009;Jiao et al 2010;Lu et al 2011). We, therefore, knocked down hDcp2 in order to determine whether it was involved in the decay of ARCA-Luc-SL.…”

“…The mRNAs are incorporated into polysomes, efficiently translated within 15 min, and both their translation and degradation rates are sensitive to the nature of the cap and the poly(A) tract. The second is the development of cap analogs that cannot be cleaved by the catalytic subunit of the human Dcp1-Dcp2 decapping complex, hDcp2 (Grudzien-Nogalska et al 2007b;Kowalska et al 2008;Su et al 2011). By blocking 59/39 degradation with a cleavage-resistant cap, one can observe 39/59 degradation in isolation.…”